PRODUCTION OF BIOETHANOL FROM TAPIOCA STARCH USING

Saccharomyces cerevisiae: EFFECTS OF TEMPERATURE AND AGITATION

SPEED

MUHAMAD FAUZI BIN IBRAHIM

A thesis submitted in fulfillment

of the requirements for the award of the degree of

Bachelor of Chemical Engineering (Biotechnology)

Faculty of Chemical & Natural Resources Engineering

Universiti Malaysia Pahang

April 2009

v

ABSTRACT

Production of bioethanol from tapioca starch involves two processes which

are enzymatic hydrolysis and fermentation. The objective of the present study was to

investigate the influences of temperature and agitation speed on the production of

bioethanol from tapioca starch using Saccharomyces cerevisiae. The fermentation

was conducted under various temperatures (30, 35 and 37°C) and agitation speeds

(100, 200 and 300 rpm) in 250 mL shake flask. The cell density, glucose

consumption and ethanol concentration were analyzed. The ethanol concentration in

the fermentation broth increased rapidly with the increased of temperature and

agitation speed. The high temperature resulted in the higher cell density and higher

glucose consumption. The high agitation speeds also preferred for both cell density

and glucose consumption. The maximum ethanol concentration of 57.8 mg/L was

obtained at a temperature of 35°C and 200 rpm of agitation speed.

vi

ABSTRAK

Penghasilan bioetanol daripada kanji ubi kayu melibatkan dua proses iaitu

hidrolisis dan penampaian. Objektif bagi kajian ini adalah untuk mengenalpasti

pengaruh suhu dan halaju adukan dalam penghasilan bioetanol daripada kanji ubi

kayu mengunakan Saccharomyces cerevisiae. Proses penapaian dijalankan pada

pelbagai keadaan suhu (30, 35 and 37°C) dan halaju adukan (100, 200 and 300 rpm)

di dalam kelalang 250 mL. Kepadatan sel, pengunaan glukosa dan kepekatan etanol

di analisa. Kepekatan etanol meningkat dengan ketara dengan peningkatan suhu dan

halaju adukan. Kenaikan suhu penampaian menyebabkan peningkatan kepadatan sel

dan peningkatan penggunaan glukosa. Halaju adukan yang tinggi pula adalah lebih

sesuai untuk penghasilan kepadatan sel dan penggunaan glukosa. Kepekatan etanol

paling maksimum adalah 57.8 mg/L diperolehi pada suhu 35°C dan halaju adukan

200 rpm.

vii

TABLE OF CONTENTS

CHAPTER ITEM PAGE

TITLE PAGE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF FIGURES x

LIST OF TABLES xi

LIST OF SYMBOLS / ABBREVIATIONS xii

LIST OF APPENDICES xiii

1 INTRODUCTION

1.1 Background of Study 1

1.2 Objectives 2

1.3 Scope of Study 2

1.4 Problem Statement 3

2 LITERATURE REVIEW

2.1 Overview of Ethanol 4

2.2 Ethanol Use as a Fuel 6

2.3 Economics and Environmental Impact 7

of Ethanol Production

2.4 Enzymes

viii

2.4.1 α-Amylase 8

2.4.2 Glucoamylases 8

2.5 Saccharomyces cerevisiae 8

2.6 Starch 9

2.6.1 Starch Composition and Structure 9

of Components

2.7 Enzymatic Hydrolysis 11

2.8 Fermentation 11

2.8.1 Effect of Temperature 12

2.82 Effect of Agitation Speed 12

3 MATERIAL & METHODOLOGY

3.1 Introduction 13

3.2 Framework of Ethanol Fermentation Process 14

3.3 Raw Materials 15

3.4 Microorganisms 15

3.5 Enzymatic Hydrolysis 15

3.5.1 Enzyme 15

3.5.2 Hydrolysis Experiment 15

3.6 Fermentation Procedures 16

3.6.1 Medium Preparation 16

3.6.2 Seed Culture Preparation 16

3.6.3 Growth Profile of Saccharomyces cerevisiae 16

3.6.4 Fermentation Study 17

3.7 Analysis Methods

3.7.1 Preparation of DNS Reagent 18

3.7.2 Total Reducing Sugar Determination 18

3.7.3 Preparation of Standard Calibration 18

Curve for Glucose

3.7.4 Ethanol Determination 19

4 RESULTS AND DISCUSSION

4.1 Introduction 20

ix

4.2 Enzymatic Hydrolysis 21

4.3 Growth profile of Saccharomyces cerevisiea 21

4.4 Ethanol Fermentation

4.4.1 Effect of different Temperature on 22

Fermentation

4.4.2 Effect of different Agitation Speed on 24

Fermentation

5 CONCLUSION AND RECOMMENDATION

5.1 Conclusion 26

5.2 Recommendation 27

REFERENCES 28

APPENDIX 32

x

LIST OF FIGURES

FIGURE TITLE

PAGE

2.1 Ethanol production costs (US$ per liter) 5

2.2 Ethanol represents closed CO2 cycle 7

2.3 Chemical structure of Amylose 10

2.4 Chemical structure of Amylopectin. 10

3.1 Framework of Ethanol Fermentation Process 14

4.1 Growth profile of Saccharomyces cerevisiea at 25°C and

150 rpm

21

4.2 The effect of temperature on cell density, glucose

consumption and ethanol concentration at constant time =

40 h for (a) 100, (b) 200 and (c) 300 rpm

23

4.3 The effect of agitation speed on cell density, glucose

consumption and ethanol concentration at constant time =

40 h for (a) 30, (b) 35 and (c) 37°C

25

xi

LIST OF TABLES

TABLE TITLE PAGE

4.1 Temperature and agitation speed in fermentation of

glucose resulted from tapioca starch 17

4.2 The composition for Standard Calibration of Glucose 19

xii

LIST OF SYMBOLS/ABBREVIATIONS

C - carbon

CO2 - carbon dioxide

DNS - Di-Nitro Salicylic Acid

g - gram

g/L - gram per liter

H - hydrogen

hr - hour

Mg2+

- ion magnesium

min - minutes

mL - milliliter

Mg - magnesium

mg - milligram

mmol - milimole

Na - sodium

OD - optical density

rpm - rotation per minute

w/v - weight per volume

% - percentage

°C - degree celsius

μL - microliter

xiii

LIST OF APPENDICES

APPENDIX TITLE

PAGE

A.1 Tapioca Starch 32

A.2 α-Amylase and Amyloglucosidase 32

A.3 Instant Yeast (Mauri-pan) 33

A.4 Shaking Water Bath (Model BS-21) 33

A.5 Double Stack Shaking Incubator Infors 33

A.6 Laminar air Flow Cabinet (Model AHC-4A1) 34

A.7 UV-Visible Single Beam Spectrophotometer (Model

U-1800)

34

A.8 Refrigerated Centrifuged (Model 5810 R) 34

A.9 Glucose hydrolyzed from tapioca starch 35

A.10 Fermentation broth before and after Fermentation 35

B.1 Data for standard calibration curve of glucose 36

B.2 Standard Calibration for Glucose 37

B.3 Data for preparation of concentration required of

ethanol standard calibration curve.

37

B.4 Graph of Standard Calibration Curve for Ethanol 38

Calculation for enzyme loading

B.5 Growth profile of Saccharomyces cerevesiae operating

at 25°C and 150 rpm for 48 hrs

38

B.6 Data of the effect of temperature and agitation speed on

ethanol fermentation.

39

B.7 Graph of peak area and data of ethanol for (35°C, 200

rpm) at retention time 4.518 min.

39

C.1 Calculation for enzyme loading 40

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Ethanol is known as ethyl alcohol or fermentation alcohol. It is referred to

one type of alcohols found in alcoholic beverages (Wyman, 2004). Due to the

unstable price and the availability of crude petroleum, the fermentation has become

an alternatives process to produce ethanol (Logsdon, 1994). Baras et al., (2002) had

reported that almost 60% of the ethanol is produced by fermentation where the major

world producers are Brazil and the US, which together account for about 80% of the

world production. Mostly ethanol produced had been widely used in cars as a fuel

alternative to gasoline.

Bioethanol is easy to manufacture and process since the raw materials used as

the feedstock is unlimited and cheaper. Major carbohydrate-containing substrates

such as cane, beet, sago and corn are used for a feedstock in ethanol production and

commonly available in tropical countries (Ramasamy et al., 2001). Starchy substrate

such as tapioca could be exploited for ethanol production. The content of tapioca

composed of 95% starch and 2% moisture. Owing to its high carbohydrate content,

tapioca becomes one of the most efficient sources of starch. This raw material has

not yet been fully exploited in highly technical industries for ethanol production.

Since the use of starch-based raw materials for ethanol production is not a common

practice, it is important to determine the optimized conditions for starch processing

in order to enhance the bioethanol utilization in Malaysia (Aggarwal et al., 2001).

2

The hydrolysis of starch may be considered as a key step in substrate

processing for bioethanol production. The main role of this step is to effectively

provide the conversion of two major starch polymer components of amylase and

amylopectin. Another crucial step would be the fermentation process that could

subsequently be converted to ethanol by yeasts or bacteria (Mojovic et al., 2006).

The parameters involve mainly pH, temperature, agitation speed, inoculum age,

medium etc. have to be evaluated and optimized in order to obtain a good yield of

bioethanol.

1.2 Objectives

The aim of this study is to determine the optimum conditions of fermentation

process for the production of bioethanol from tapioca starch. The objectives of

this research are:

To determine the effect of temperature on the production of bioethanol from

tapioca starch using Saccharomyces cerevisiae.

To determine the effect of agitation speed on the production of bioethanol

from tapioca starch using Saccharomyces cerevisiae.

1.3 Scope of the Study

Bioethanol production process has two steps which are enzymatic hydrolysis

and fermentation process. The scope for this study was to determine the yield of

bioethanol that can be produced using tapioca starch in fermentation process. The

250 mL fermentation will conducted to investigate the effects of temperature and

agitation speed in fermentation process. The optimum bioethanol production from

tapioca starch using Saccharomyces cerevisiae was aimed. Others parameters such as

pH, dissolved oxygen, nutrient and time were fixed during the process study.

3

1.4 Problem Statement

A long time ago until now, gasoline u.sages have a higher demand every

year. In recently, the world face a crisis of diminishing fossil fuel reserves, thus an

alternative energy sources need to be renewable, sustainable, efficient, cost-effective,

convenient and safe (Chum and Overend, 2001).

In June 2008, the price of Malaysia gasoline increased by 40% from

RM1.92/litre to RM2.70/litre. In order to reduce the use a large amount the gasoline

in daily, Malaysian researches and development have focused on commercially

produced ethanol as an alternative fuel. Fermentative production of ethanol from

renewable resources has received attention due to increasing petroleum shortage.

Most of the raw materials utilized for bioethanol production were corn grain and

sugar cane (Mojovic et al., 2006). It is important to see the potential of raw materials

rich in fermentable carbohydrates such as tapioca since it is largely available in

Malaysia.

Gasoline is a finite resource that cannot be sustained indefinitely, and its price

is increased as its availability is decreased over time. Gasoline can produce toxic

substances and gaseous emissions when it is combusted. It will cause the negative

impact on the environment, particularly greenhouse gas emissions and these

problems have warned the society to find another renewable fuel as an alternative.

Bioethanol on the other hand is most environmental friendly. It is known as a high

octane fuel with lower emissions can be use in car as fuel. It can be produced using

cheaper materials biologically for feedstock and is already compatible, in low blends,

with existing gas engines (Wyman, 2004).

4

CHAPTER 2

LITERATURE REVIEW

2.1 Overview of Ethanol

In recent years, ethanol is one of the most important renewable fuels

contributing to the reduction of negative environmental impacts generated by the

worldwide utilization of fossil fuels (Cardona and Sanchez, 2007). Due to the

diminishing fossil fuel reserve, research and development efforts directed toward

commercial production of ethanol from renewable resources have increased

(Mojovic et al., 2006).

Brazil and the United States lead the industrial world in global ethanol

production, accounting together for 70% of the world's production and nearly 90% of

ethanol is used for fuel. In 2006, Brazil has produced 16.3 billion liters of ethanol

represents 33.3% of the world's total ethanol production. Sugar cane plantations

cover 3.6 million hectares of land for ethanol production with a productivity of 7,500

liters of ethanol per hectare. The U.S. in the other hand, 3,000 liters per hectare of

maize ethanol was produced (Cardona and Sanchez, 2007).

Fermentation alcohol has been investigated for several years. Substrate used

for this process essentially depends on surplus grains production of each country. In

the United States maize was used whereas, in France, the principal cereal for alcohol

production is wheat. Several studies on alcohol have been carried from cellulosic

biomass, cassava, sago, sorghum, blackstrap molasses, and maize but few on raw

5

wheat flour as substrate. Figure 2.1 shows the countries that have the worlds least

cost of ethanol production.

Figure 2.1: Ethanol production costs (US$ per liter) (Salomao, 2005)

In daily application, ethanol is mostly used as fuels (92%), industrial solvents

and chemicals (4%) and beverages (4%) (Logsdon, 2006). An important issue

regarding the ethanol production is weather the process is economical. Research

efforts are focused to design and improve a process, which would produce a

sustainable transportation fuel. A low cost of feedstock is a very important factor in

establishing a cost effective technology (Mojovic et al., 2006). Therefore, a strong

need exists for efficient ethanol production with low cost raw material and

production process (Liu, 2007).

6

2.2 Ethanol Use as a Fuel

Ethanol is known as ethyl alcohol or fermentation alcohol, often referred to as

just ‗‗alcohol,‘ and has the chemical formula of C2H5OH. It is a colorless, clear

liquid that looks like water and is completely miscible with water. Ethanol has a

somewhat sweet flavor when diluted with water; a more pungent, burning taste when

concentrated; and an agreeable ether-like odor. Other that, it is more volatile than

water, flammable, burns with a light blue flame, and has excellent fuel properties for

spark ignition internal combustion engines (Wyman, 2004).

Ethanol has decreased the need for other octane booster such as benzene,

which are toxic and often carcinogenic. The oxygen in ethanol reduces emissions of

burned hydrocarbons and carbon monoxide, particularly for older vehicles that tend

to burn rich (Wyman, 2004). As an oxygenated compound, ethanol provides

additional oxygen in combustion, and hence obtains better combustion efficiency.

Since the completeness of combustion is increased by the present of oxygenated

fuels, the emission of carbon monoxide is reduced by 32.5% while the emission of

hydrocarbon is decreased by 14.5% (Rasskazchikova et al., 2004). Nguyen et al.,

(1996) has stated the ethanol has lower environmental impact than petrol in terms of

the combustion by-products and greenhouse gases.

The use of ethanol as fuel goes back to origin of the use of vehicles itself. For

example, Henry Ford‘s Model T. built in 1908, ran on ethanol. It was continued until

the availability of cheap petrol effectively killed off ethanol as a major transport fuel

in the early part of the 20th century. The energy crisis of the 1970s then renewed

interest in ethanol production for fuels and chemicals (Marrs, 1975).

7

2.3 Economics and Environmental Impact of Ethanol Production

Ethanol made from biomass provides unique environmental, economic

strategic benefits and can be considered as a safe and cleanest liquid fuel alternative

to fossil fuels. The ethanol yields and process economics along with the technical

maturity and environmental benefits are the key parameters that determine the

feasibility of bioethanol production (Nguyen and Saddler, 1991). The use of

bioethanol as a source of energy would be more than just complementing for solar,

wind and other intermittent renewable energy sources in the long run (Lin and

Tanaka., 2006).

Ethanol has provided multiple economic, social and environmental benefits to

the producing regions and to the country as a whole. Ethanol has made a significant

contribution to the reduction of greenhouse gas emissions, substituting ethanol for

gasoline for fossil fuels (Lucon et al., 2005). Furthermore, ethanol represents closed

carbon dioxide cycle because after burning of ethanol, the released carbon dioxide is

recycled back into plant material because plants use CO2 to synthesize cellulose

during photosynthesis cycle (Wyman, 2004). Ethanol production process only uses

energy from renewable energy sources. Hence, no net carbon dioxide is added to the

atmosphere, making ethanol an environmentally beneficial energy source (Figure

2.2).

Figure 2.2 Ethanol represents closed CO2 cycle (Chandel et al., 2007).

8

2.4 Enzymes

2.4.1 α-Amylases.

α-Amylasesare obtained from Aspergillus oryzae, Bacillus amyloliquefaciens,

and Bacillus licheniformis are mostly used in starch hydrolysis for sugar syrups and

brewing. The end products from the action of a-amylase on starch are glucose,

maltose, maltotriose. maltotetraose. maltopentaose, and maltohexaose (Nigam and

Singh., 1994)

2.4.2 Glucoamylases.

Glucoamylases obtained Aspergillus niger and Rhizopus species are used in

glucose syrup production from liquefied starch (Nigam and Singh., 1994).

2.5 Saccharomyces cerevisiae

Saccharomyces cerevisiae (brewer‘s and baker‘s yeast) has been used in

classical food fermentation, the production of fuel alcohol, glycerol, invertase and

animal feeding (Camacho-Ruiz et al., 2003). Saccharomyces cerevisiae is found in

the wild growing on the skins of grapes and other fruits. There are two ways

Saccharomyces cerevisiae breaks down glucose which are aerobic and anaerobic

conditions. Saccharomyces cerevisiae strains for ethanol production due to its high

ethanol yield and high tolerance to rather high ethanol concentration.

9

2.6 Starch

Starch, chemical formula (C6H10O5)n is a polysaccharide carbohydrate

consisting of a large number of glucose units joined together by glycosidic bonds. It

is consisting of amylase (linear chain of glucose) and amylopectic (branched chain of

glucose). Starches are found in a large number of plants as the major carbohydrates

reserve and provide an essential source of energy. The common source of starch

comes from maize, wheat, potatoes, tapioca and rice (Marrs, 1975).

Starch of tapioca is a major starch sources and has a wide commercial use. A

typical composition of the tapioca root is moisture (70%), starch (24%), fiber (2%),

protein (1%) and other substances including minerals (3%). Tapioca ranks very high

among crops that convert the greatest amount of solar energy into soluble

carbohydrates per unit of area. Among the starchy staples, tapioca gives a

carbohydrate production which is about 40% higher than rice and 25 % more than

maize. Besides, tapioca is cheapest sources of calories for both human nutrition and

animal feeding (Nyerhovwo, 2004)

Tapioca starch, an agricultural product, is a cheap substrate that easily

available in tropical countries (Ramasamy et al., 2001).Tapioca starch can be

isolated in a pure form with least contamination of non starchy constituents.

Generally in industrial application, tapioca starch is used in paper, pharmaceutical

and textiles industries as well as in manufacture of explosion adhesives and baby

food (Kunamneni and Singh. 2005). The use of tapioca as a source of ethanol for fuel

is already being exploited and found to be very promising (Nyerhovwo, 2004).

2.6.1 Starch Composition and Structure of Components

Amylose consists of anhydroglucose units that are linked by α-D-1, 4

glucoside bonds to form linear chains (Figure 2.3). Amylose molecules are typically

made up from 200-2000 anhydroglucose units. The level of amylose and its

10

molecular weight vary between different starches types. Aqueous solutions of

amylose are unstable due to intermolecular attraction and association of neighboring

amylose molecules. This leads to viscosity increase, retrogradation and precipitation

of amylose particles (Hedley, 2002).

Figure 2.3: Chemical structure of Amylose. (Reis et al., 2002).

Amylopectin have a polymeric, branched structure (Figure 2.4).. Its consist of

anhydroglucose units that are linked by α -D-1,6 bonds that occur every 20-30

anhydroglucose units. In addition, it also have α-D-1,4 bonds that are present in

amylose.The level of amylopectin varies between different starches types. Aqueous

solutions of amylopectin are characterized by high viscosity, clarity, stability and

resistance to gelling (Hedley, 2002).

Figure 2.4: Chemical structure of amylopectin. (Reis et al., 2002).

11

2.7 Enzymatic Hydrolysis

There are two major processes of converting rich fermentable carbohydrates

materials to ethanol which are enzymatic hydrolysis (from carbohydrates to sugars)

and fermentation by microorganisms (from sugars to ethanol). The main role of

enzymatic hydrolysis is to effectively provide the conversion of two major starch

polymer components (amylose and amylopectin) to fermentable sugar that could

subsequently converted to ethanol by yeast (Aggarwal et al., 2001). The degree of

hydrolysis of native starch depends on the factors such as the substrate concentration,

type and concentration of enzyme used, and on the applied process conditions such

as pH, temperature and the mixing rate (Mojovic et al., 2006).

There are two processes in enzymatic hydrolysis; liquefaction and

saccharification. The breakdown of large particles drastically reduces the viscosity of

gelatinized starch solution, resulting in a process called liquefaction. The final stages

of depolymerization are mainly the formation of mono-, di- , and tri-saccharides.

This process is called saccharification (Barsby et al., 2003).

2.8 Fermentation

The generation of energy without the electron transport chain is called

fermentation. This definition is the exact and original meaning of the term

fermentation (Shuler and Kargi, 2002). Yeast convert glucose to ethanol and carbon

dioxide fermentation, as shown by the following reaction:

C6H12O62C2H5OH + 2CO2 (Equation 2.1)

Fermentation can be performed as a batch, fed batch or continuous process.

The choice of most suitable process will depend upon the properties of

microorganisms and type of starch (Chandel et al., 2007). There are many parameters

12

should be concerned in fermentation process such as temperature, agitation speed,

pH value, dissolved oxygen and nutrient. The effect of temperature and agitation

speed on ethanol production is important for the successful progress of the

fermentation.

2.8.1 Effect of Temperature

Temperature has a marked influence on the production of ethanol. According

to Rivera et al., (2006) suitable temperature in fermentation process is the good

condition for the yeast to react. Too high temperature kills yeast, and low

temperature slows down yeast activity. Thus, to keep a specific range of temperature

is required. Normally ethanol fermentation is conducted at temperature range

between 30-35°C which stated by Shuler and Kargi, (2002) that the ethanol will be

produced at highest concentration.

2.8.2 Effect of Agitation Speed

Agitation is important for adequate mixing, mass transfer and heat transfer. It

assists mass transfer between the different phases present in the culture, also

maintains homogeneous chemical and physical conditions in the culture by

continuous mixing. Agitation creates shear forces, which affect microorganisms,

causing morphological changes, variation in their growth and product formation and

also damaging the cell structure (Kongkiattikajorn et al., 2007).

CHAPTER 3

MATERIALS & METHODOLOGY

3.1 Introduction

This chapter will discuss about the process for producing ethanol through

fermentation process. In this study, bioethanol was produced while varying the effect

of temperature and agitation speed during the fermentation processes. Meanwhile in

enzymatic hydrolysis, liquefaction and saccharification processes are involved. The

glucose consumption was observed by DNS method and the concentration of ethanol

was determined using gas chromatography. The flowchart of the process is shown in

Figure 3.1.

14

3.2 Framework of Ethanol Fermentation Process

Raw Material

(Tapioca Starch)

Liquefaction

Saccharafication

Growth Profile

Fermentation

(Various of Temperature and

Agitation Speed)

Cell, Glucose and Ethanol

Analysis

Seed Culture

Medium Preparation

Figure 3.1: Framework of Ethanol Fermentation Process

15

3.3 Raw Materials

Starch from tapioca flour is utilized as starchy substance for the production of

sugar.

3.4 Microorganisms

Saccharomyces cerevisiae is used as microorganism in bioethanol

fermentation of tapioca starch.

3.5 Enzymatic Hydrolysis

3.5.1 Enzymes

α-Amylase from Bacillus licheniformis was used for liquefaction process.

The enzyme activity was 144 KNU mL-1

(KNU, kilo novo units α-amylases— the

amount of enzyme which breaks down 5.26 g of starch per hr according to

Novozyme‘s standard method for the determination of α-amylase). Meanwhile, for

the glucoamylase employed, its initial activity is 240 AGU/g (AGU is the amount of

enzyme which hydrolyses 1 mmol of maltose per minute under specified conditions).

3.5.2 Hydrolysis Experiment

Tapioca starch, 100 g was mixed with water to get 33% w/v concentration.

The mixture was hydrolyzed with enzymes (α-Amylase and glucoamylase) in two

steps. Initially, the slurry of tapioca flour was liquefied with 80 μL of α-amylase at

85°C and pH 6.0 for 1 hr in water bath with 150 rpm agitation speed. Then,